COMBINED Slides MMA Program... · • MMA can be used as a secondary crossmatch technique for patients with unusual antibodies We connect donors and the lives they impact. Interp

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2019 Advanced Track Webinars

Link to register: https://immucor.webinato.com/register

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learn.immucor.com

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Continuing Education

• PACE, Florida and California DHS

• 1.0 Contact Hours

• Each attendee must register to receive CE at: https://www.surveymonkey.com/r/AdvancedAntibodyWork-Ups

• Registration deadline is March 22, 2019

• Certificates will be sent via email only to those who have registered April 5, 2019


Questions?

• You are all muted

• Q&A following session - Type in questions


• Course content is for information and illustration purposes only. Immucor makes no representation or warranties about the accuracy or reliability of the information presented, and this information is not to be used for clinical or maintenance evaluations.

• The opinions contained in this presentation are those of the presenter and do not necessarily reflect those of Immucor.

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Katrina Billingsley, MSTM, MT(ASCP)SBB

Advanced Antibody Work-Up Techniques: MMA & Molecular Testing

We connect donors and the lives they impact.

Objectives

Upon completion of this lecture, the attendee will be able to:• Define Monocyte Monolayer Assay (MMA) and determine

when it should be considered• Understand the role of molecular testing in the blood

bank• Evaluate the application of MMA and molecular testing

using a case study approach


MMAAKA – your patient has an anti-WHAT…and you need HOW many units of blood?

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What is MMA?

• An in vitro assay used to predict the clinical significance of red cell antibodies– Transfusion candidates

• Antibody to high frequency antigen (known/unknown)• Multiple alloantibodies

– HDFN evaluation


What is clinical significance?

Clinically significant RBC antibodies are defined as “those associated with HDFN, hemolytic transfusion reactions or notably decreased survival of transfused red cells.”

– AABB Technical Manual. 19th Ed. pg461


Factors related to the antibody

• Binding constant • Ig class and subclass• Ability to bind to macrophage Fc receptors• Ability to activate complement• Thermal reactivity range• Plasma concentration

red blood cell

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Factors related to the antigen

• Antigenic determinant “epitope”

• Abundance of sites on the RBC

• Distribution in the body• Association with complement

activationeclinpath.com


Factors affecting the Ag/Ab bond

• Spatial complementarity between antigen and antibody: “Lock and Key” concept

• Weak non-specific intermolecular forces including: electrostatic charges (ionic groups), hydrogen bonds, hydrophobic (non-polar) bonds, Van der Waals forces

• The equilibrium (association) constant of the Ag-Ab formation


Factors related to the RES

• Fc receptor polymorphism

• Phagocytic activity of the mononuclear phagocytic system

• Diagnosis

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• One element missing in the pathogenesis process can result in normal survival of antigen positive RBCs

• MMA can be used as a secondary crossmatch technique for patients with unusual antibodies


Interp. based on definition of “significance”

Arndt PA, Garratty G. Transfusion 2004;44:1273-81

If significance = abnormal RBC survival

• MMA may not predict abnormal T50Cr• Most AHG antibodies to high incidence antigens would be

classified as clinically significant.

If significance = laboratory signs without clinical symptoms of a reaction

If MMA = Then:

≤5% 20% had only laboratory signs of a reaction.

5.1-20% 67% had only lab signs of a reaction.

>20% 75% had only laboratory signs of a reaction.

If significance = a clinically obvious reaction

If MMA = Then:

≤5% Incompatible blood could be given with little risk.

5.1-20% 33% of patients had clinical signs of a reaction

>20% 64% of patients had clinical signs of a reaction.


How does it work?

• Mononuclear cells have specific receptors for IgG, IgM and the C3 component of complement

• Receptors bind to the Fc portion of antibodies

• Antibody coated RBCs can adhere to and be ingested by the macrophages

ROBERT TEMPKIN

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Separation of Monocytes Preparation of Monolayer


Sensitization of RBCs Addition of RBCs to Monolayer


Counting the Monocytes

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DNAAKA – your patient has an anti-WHAT…and you need this genotype when?


What is genotyping?

• The process of determining differences in the genetic make-up of an individual’s DNA sequence using biological assays and comparing it to a reference sequence…

• Identify genetic variations


Single Nucleotide Polymorphism (SNP)

• Change of a single base pair in the triplet codon

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Who do we genotype?

• Donors– based on patient

demographic served– screen for “rares”– in-house reagent red

cell preparation

• Patients– Prophylactic matching

for chronic transfusion– Post transfusion samples

• Eliminate need for special techniques

– Antibody ID aid• High incident antigens


Case Studies


Patient SD

• 19 y/o African American female• Placenta Previa, active bleeding

– 3 units emergently transfused

• Prenatal antibody screen negative• Currently all panel cells positive• Sample sent to genotyping lab

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PreciseType HEA genotype by Immucor


Patient SD

• Anti-U, -Jka, -E identified• Anti-U & anti-Jka detected in cord blood eluate

– Which antibody is causing HDFN?

• Would baby need exchange?• MMA to predict most clinically significant antibody


Patient SD

AntibodySpecificity

IAT MI %

U 3+ 32

Jka 2+ 9.2

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Patient SD

• Although MI >5, anti-Jka appears less significant than anti-U

• Antibody is self-limiting• E-, U-, Jk(a+) unit recommended if exchange

required


Patient LD

• 12 y/o African American female, SCD• O, Rh positive, aby screen pos• 2005 – 2010

– anti-Fya, M, -V, -He, -Lea, -Leb, cold & warm auto

• 2011 – anti-D, -e-like– Pt is R1r– allo or auto?


Patient LD

• MMA request– Can M+ units be used for transfusion

• Genotype request– Evaluation of RHD and RHCE gene for variant status

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Patient LD MMA 1

RBC Phenotype IAT MI (%)

Phenotype similar 1+ 0

M+ 1+ 0.2

M+ 1+ 0

One-stage method


Patient LD MMA 1


Phenotype similar 1+ 0.5

M+ 1+ 0.5

M+ 1+ 50.7

Two-stage Complement method


Patient LD MMA 1


Phenotype similar 1+ 0.5

M+, Le(a-b-) 1+ 0.5

M+, Le(a-b+) 1+ 50.7


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Patient LD

• Majority of MI comprised of adhered cells

• Was the anti-Leb causing the clinically significant result in the presence of C’?


Patient LD MMA 2


Le(a-b-) 1+ 0

Le(a-b+) 1+ 52.9

Le(a+b-) 1+ 3.5



Patient LD

• Le(b-) units recommended for transfusion

• Genotyping on BioArray RHD and RHCE– No variant detected– Antibody Rh specificity likely part of auto

• Pt now receiving Rh D+ units– Increased availability of donors

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Patient EH

• 62 year old African American female• History of previous transfusion• Acute pancreatitis• Hgb 3.3 g/dL


Patient EH

• Serology– Antibody reacts 1-3+– Autocontrol negative– D-- cells negative

• MMA• Complete genotype• RHCE genotype


Patient EH – MMA

RBC SourceAdhered

RBCs (%)Ingested

RBCs (%)Total (%)

RO 0.00 0.00 0.00

RO 1.00 0.00 1.00

rr 0.25 0.00 0.25

rr 0.00 0.25 0.25

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Patient EH

• Complete genotype

• No variant or nulls detected

C E c e K Fya Fyb Jka Jkb M N S s

0 0 + + 0 0 0 + + 0 0 0 +


Patient EH

• RHCE genotype– RHCE*ceAR/RHCE*ceEK– Hr-, hrS-

• Additional serology confirmed presence of both antibodies


Patient EH

• Patient has anti-Hr with anti-hrS• Clinical significance

– No to fatal

• Two units emergently transfused• Repeat MMA if additional transfusion needed

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Patient PK

• 20 y/o AA female• Sickle Crisis• Hospital ID anti-e

– Phenotype

– Presumptive aby ID – “little-e like”– Transfused 3 R2R2 units

C E c e

0 0 + +


Patient PK

• 3 weeks later…• Anti-E now present• Sample sent to LBC

– Monocyte Monolayer Assay– RHCE genotype


Patient PK

• RHCE genotype− RHCE*ce / RHD*r’s-RHCE*ce733G,1006T− Appears to have one WT and one variant allele− No anti-hrB− Reactivity likely due to warm auto

• What if the geno is incomplete…− Cases reported of aby production in spite of WT allele

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Patient PK

RBC Source AHG% Adhered

RBCs% Ingested

RBCs% Total MI

Pheno Matched, hetRHCE*ce733G,1006T

0 0.50 9.50 10.00

Pheno SimilarhrB+

mi+ 0 28.75 28.75

Pheno SimilarhrB+

mi+ 0 30.00 30.00

Pheno SimilarhrB+

mi+ 0.25 24.75 25.00

Pre-transfusion A/C 0 0.25 13.50 13.75


Patient JM

• 66 y/o Hispanic male• History of anti-Ge3• Current diagnosis: septic shock


Anti-Ge3

• IgG more common than IgM• Complement binding• Hemolytic• Clinical significance

– No to moderate, immediate or delayed

Blood Group Antigen Factsbook, 3rd edition

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Patient JM


Ge:3+ 4+ 76.75

Ge:3+ 4+ 83.00

Ge:3+ 4+ 84.75

Ge:3+ 4+ 80.50

Ge:3+ 4+ 52.50


Patient JM

• Hospital disregarded recommendation• Initiated transfusion• Transfusion immediately discontinued as patient

exhibited signs of acute hemolytic transfusion reaction


Conclusion

• A positive crossmatch means:– An agglutinin (antibody) is present in the serum– RBCs express the corresponding antigen

• A clinically significant antibody is one capable of causing a notable decrease of the survival of antigen positive RBCs and/or hemolytic reaction

• DNA and MMA can aid in ABID and the selection of blood

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g{tÇ~ lÉâ4Katrina Billingsley, MSTM, MT(ASCP)SBBManager, Scientific Support ServicesLifeShare Blood CenterShreveport, LA 71106318-673-1546

[email protected]


Questions?



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Questions?




Continuing Education

• PACE, Florida and California DHS

• 1.0 Contact Hours

• Each attendee must register to receive CE at: https://www.surveymonkey.com/r/AdvancedAntibodyWork-Ups

• Registration deadline is March 22, 2019

• Certificates will be sent via email only to those who have registered April 5, 2019

2/18/2019

22


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Documents

COMBINED Slides MMA Program... · • MMA can be used as a secondary crossmatch technique for patients with unusual antibodies We connect donors and the lives they impact. Interp